臺灣博碩士論文加值系統

自體螢光光譜的發現及其應用，包括癌症的早期判定、老年癡呆症的檢測和動脈硬化辨別，其利用非侵入式的檢測能有效降低侵入式檢測造成的傷害。其中主要的鑑別是在於自體螢光物質—膠原蛋白(collagen)、啶黃素(flavins)和煙醯胺腺嘌呤二核苷酸(NADH)在生物體中的變化，目前的技術已經可以將螢光物質的變化和特徵應用在病理的判讀上。
NADH是電子傳遞鏈中的供給者，作為生物體獲得能量的重要一環，並可以做為生物能量需求研究的指標，螢光生命週期顯微術 (FLIM) 便是其中最靈敏的技術之一。FLIM基於測量螢光分子在激發態中所花的平均時間不同，而獲得實驗參數進而研究深入的動態過程。螢光生命週期提供了解細胞的構造變化、折射率改變，黏滯系數和pH值等。在自體螢光NADH的應用中，FLIM的量測提供了自由與蛋白質結合態的比例差異，藉由此一非侵入式量測，提供量測細胞代謝的方法。由NADH結合的粒線體內膜蛋白所參與的複雜化學反應，產生三磷酸腺苷(ATP)為細胞中主要的能量儲存方式。由此我們可以藉由量測NADH作為了解細胞代謝的能量需求。
一般螢光生命週期可以獲得0.4 ns的自由態與2.5 ~ 3 ns 蛋白質結合態。與先前的方式做比較，此一方式可以提供良好的解析度。然而在蛋白質結合態中具有與自由態相近的短生命週期，無法清楚分辨。
藉由偏振的入射光可以提供異向性的排列形式，螢光的放光會因結合的重量不同而有不同的旋轉角度，因此量測垂直與平行的極化偏振光強作比較，並計算異向性的數值，便可以深入了解自由與蛋白質結合態在細胞中的比例差異。
本實驗由原本的FLIM系統升級，藉由加裝的偏振片讓我們可以偵測螢光的異向性。應用該系統於NADH的結合與自由態比例量測。並由細胞在不同階段的細胞培養增長，使我們獲得自由與結合態的差異。我們的研究結果清楚地表明，異向性的數值從最3天的緩緩下降到第4天開始增長至最後，並且在細胞培養群聚的邊緣與中心的細胞群聚有差異，這個結果與先前利用FLIM所量測的結果吻合。

In this study, detection is constructed and integrated onto two-photon FLIM system. Specialized software is also developed to analyze the anisotropy data recorded. The system is then applied for the characterization of the free/bound ratio of NADH in cells at different stages of cell culture growrh.
One of the major intrinsic fluorophores, nicotinamide adenine dinucleotide reduced (NADH) is a principal electron donor and transporter both in glycolisis and oxidative phosphorylation. The discovery of its fluorescence properties by Chance in 50s led to the establishment of new field – bioenergetics, which studies the turnover of energy in cells. This work brought in a surge of the fluorescence spectroscopy applications for the observations of processes in mitochondria in vitro, and, as the technologies improved, in vivo.
One of the most powerful techniques currently applied in non-invasive bioenergetics studies is fluorescence lifetime imaging microscopy (FLIM). FLIM measures the average time fluorescent molecules stayed on the excited states. Observing the changes in fluorescence lifetime provides insights on a broad range of molecular dynamic processes, including conformation, changes in refractive index, viscosity and pH of the environment, etc. In contrast with the biochemical analysis, which requires extraction of pyridine nucleotides, fluorescence lifetime is able to assess the free/bound ratio of NADH, in vivo and non-invasively, thus providing an insight into the metabolic activity of a cell. When bound to the dehydrogenases of Complex I in mitochondrial inner membrane, NADH participates the synthesis of adenosine triphosphate (ATP), the major energy repository and transporter in cell. Thus the increasing of the bound NADH quantities is associated with the increased metabolic activities.
The generally applied two-component model (0.4 ns for free and ~2.5-3ns for bound NADH) is, nevertheless, a simplification, which applies well to a broad variety of situations. However, detailed studies are required to futher elucidate the dynamics. The differentiation between free and bound forms can be conducted by polarization microscopy. If a fluorophore is excited by polarized light, the initial fluorescence will be predominantly polarized in parallel to the excitation light, i.e., the fluorescence is emitted anisotropically. However, subsequent rotational diffusion of the fluorophores will result in a randomization of orientation. Thus, measurement of the polarization anisotropy can provide an insight on the rotational diffusion and hence allow the discrimination between the free and the bound forms. Due to the added complexity this approach is currently used by selected few groups.
Our results clearly demonstrated that a decrease in the free/bound ratio starting from the 4th day of the cell culture growth as it proceeds from the initial to the exponential growth stage. These results agree well with and may provide better contrast than the data obtained earlier by pure FLIM studies.

論文電子檔著作權授權書 I
論文審定同意書 II
誌謝 III
摘要 V
ABSTRACT VII
目錄 IX
表目錄 XIII
第一章 簡介 1
第二章 實驗原理 4
2-1. 細胞的生理代謝 4
2-2. 自發螢光 6
2-3. 時間解析螢光生命週期影像 8
2-4. 螢光分子的偏振與異相性 10
2-5. 實驗方法 13
第三章 螢光生命週期量測結果 15
第四章 實驗架設與技術 20
4-1. 實驗系統架設 20
4-2. 時間解析單光子計數儀(TCSPC) 22
4-3. 數據分析 23
4-4. 樣品製備 24
4-5. 系統校正與測試 25
第五章 實驗結果 26
5-1. 螢光異相性量測 26
5-2. 群集邊緣與中心的比較 29
5-3. 垂直與平行訊號的比較 32
第六章 討論 34
6-1. 螢光異相性與生命週期的比較 34
6-2. 螢光異向性與細胞培養時間 35
6-3. 推論 35
6-4. 結論 36
第七章 未來展望 38
第八章 參考資料 40

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